表达绿色荧光蛋白的MVA重组毒株的构建及筛选

为了构建及筛选表达绿色荧光蛋白（GFP）的改良型痘苗病毒安卡拉（MVA）重组毒株，并对其进行鉴定，本研究基于同源重组原理设计引物，通过PCR扩增获取含有MVA两侧同源臂的外源基因GFP片段，将GFP基因片段转染到感染了MVA的CEF细胞中使之同源重组到MVA ORF086-087位点（基因组中70303-70304 bp之间），利用倒置荧光显微镜观察并标记表达GFP的单个噬斑，筛选获取重组毒株，应用倒置荧光技术、PCR及Western blotting对该重组毒株进行鉴定。结果显示，经过3轮噬斑筛选，在倒置荧光显微镜下可观察到大量表达GFP的单个噬斑，PCR扩增检测结果表明目的基因已成功整合到重组毒株MVA-GFP中。Western blotting结果表明，GFP在感染的细胞内成功表达。本研究利用基因工程技术成功获得表达GFP的重组毒株MVA-GFP，可为进一步将其他抗原基因插入GFP位点中筛选无标记的重组毒株及疫苗研究提供材料。

Construction and Screening of GFP Recombinant MVA Strain

To construct and screen the recombinant modified vaccinia virus Ankara (MVA) strain expressing green fluorescent protein (GFP),primers were designed and synthesized.GFP gene was amplified by PCR and inserted into the ORF086-087 site (70303-70304 bp) of MVA based on the principle of homologous recombination.Fluorescence microscope was used to select single plaque with GFP as the reporter gene,and the recombinant strain was identified by fluorescence technique,PCR and Western blotting.The results showed that a large number of single plaques expressing GFP could be observed under fluorescence microscope,GFP gene successfully intergrated into recombinant strain MVA-GFP genome.Western blotting result showed that GFP was successfully expressed in infected cell.In this study,the recombinant strain MVA-GFP expressing GFP was constructed by genetic engineering technology,which provided materials for further screening unmarked recombinant strain and vaccines by inserting foreign target genes into GFP site.